TAILIEUCHUNG - Báo cáo khoa học: Phage-display as a tool for quantifying protein stability determinants

To address questions of protein stability, researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phage-displayhasprovedtobeapowerful tool foranalyzingprotein stabilitydue to the large library sizeand the robustnessof the phageparticle the B1 domain of protein G (GB1) and a camelid heavy chain antibody as model systems, we are using phage-display lib-raries toexperimentallyaddressquestions thathavegenerally been addressedin silico, either through computational stud-ies or statistical analysis of known protein structures | Eur. J. Biochem. 271 1623-1629 2004 FEBS 2004 doi MINIREVIEW Phage-display as a tool for quantifying protein stability determinants Joanne D. Kotz1 Christopher J. Bond2 and Andrea G. Cochran1 1 Department of Protein Engineering and 2Medicinal Chemistry Genentech Inc. South San Francisco CA USA To address questions of protein stability researchers have increasingly turned to combinatorial approaches that permit the rapid analysis of libraries of protein variants. Phagedisplay has proved to be a powerful tool for analyzing protein stability due to the large library size and the robustness of the phage particle to a variety of denaturing conditions. With the B1 domain of protein G GB1 and a camelid heavy chain antibody as model systems we are using phage-display libraries to experimentally address questions that have generally been addressed in silico either through computational studies or statistical analysis of known protein structures. One effort has focused on identifying novel solutions to repacking the hydrophobic core of GB1 while maintaining stability comparable to the wild type protein. In a second study a small set of substitutions in complimentarity-determining region 3 was found to stabilize the framework of the camelid antibody. Another major focus has been to obtain quantitative data on b-sheet stability determinants. We have successfully adapted a phage-display method for quantitating affinities of protein variants shotgun alanine scanning to analysis of GB1 stability. Using this method we have analyzed the energetic contributions of cross-strand side chainside chain interactions. Finally we discuss parameters to consider in using phage-display to discriminate subtle stability differences among fully folded variants. Overall this method provides a fast approach for quantitatively addressing biophysical questions. Keywords beta sheet hydrophobic core phage-display protein G protein stability. Introduction Understanding .

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